At present, functional analysis of proteins has become important as post-genome-related technology in medical care, drug development, and food industry. Particularly, in order to analyze cellular action, it is necessary to investigate interaction (binding, separation) between a protein as a biological substance and another protein or a low-molecular compound in a living cell.
The interaction between a protein as a biological substance and another protein or a low-molecular compound in a living cell is analyzed by utilizing a fluorescence resonance energy transfer (FRET) phenomenon. Interaction between molecules in a region of several nanometers can be measured by measuring fluorescence generated by the FRET phenomenon. FRET refers to a phenomenon in which, when a donor fluorescent substance is excited by laser light irradiation, part of excitation energy is transferred to an acceptor fluorescent substance located close to the donor fluorescent substance without emitting fluorescence so that the acceptor fluorescent substance emits fluorescence.
When the presence or absence of the occurrence of FRET is investigated by giving a fluorescent substance to a biological substance such as a protein, a method is conventionally used in which the presence or absence of the occurrence of FRET is investigated based on a change in the intensity of fluorescence emitted from the fluorescent substance irradiated with laser light. More specifically, this method measures the decrement of the fluorescence intensity of donor fluorescence emitted from a donor fluorescent substance due to the transfer of part of excitation energy from the donor fluorescent substance and the increment of fluorescence intensity due to emission of acceptor fluorescence from an acceptor fluorescent substance using the transferred excitation energy. However, this method cannot always accurately judge the presence or absence of the occurrence of FRET because the decrement and the increment vary depending on the amount of the donor fluorescent substance or the acceptor fluorescent substance (label) contained in a measuring object.
On the other hand, as a method less likely to be influenced by the amount of a label, such as a donor fluorescent substance or an acceptor fluorescent substance, contained in a measuring object, a method is known in which the fluorescence lifetime of donor fluorescence emitted from a donor fluorescent substance is measured, and the presence or absence of the occurrence of FRET is judged based on a change in the fluorescence lifetime (Patent Literature 1).